Use of a ppar-delta agonist for reducing loss of muscle strength, muscle mass, or type I muscle fibers in an immobilized limb

ABSTRACT

The present invention provides methods for reducing loss of muscle strength, muscle mass, or Type I muscle fibers in an immobilized limb by administering (E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]acetic acid or a pharmaceutically acceptable salt thereof.

BACKGROUND OF THE INVENTION

Field of Invention

The invention relates to the fields of pharmacology and medicine, andprovides therapeutic methods and compositions for treating muscleatrophy.

Description of Related Art

Muscle atrophy refers to the loss of muscle mass and/or to theprogressive weakening and degeneration of muscles, including theskeletal or voluntary muscles (which control movement), cardiac muscles(which control the heart), and smooth muscles. Skeletal muscle atrophyis associated with bed rest, corticosteroid use, denervation, chronicrenal failure, limb immobilization, neuromuscular disorders, sarcopeniaof aging, and arthritis. Irrespective of the underlying cause ofatrophy, reduced muscle activation/contractile activity (hypodynamia) isan invariant feature. The fundamental molecular mechanism(s) underlyingmuscle atrophy and numerous cellular processes include decreased proteinsynthesis, increased protein degradation, suppression of bioenergeticpathways associated with mitochondrial function, and increased oxidativestress (Abadi et al., PLoS ONE 4(8):e6518 (2009)).

Upstream triggers that initiate muscle atrophy are poorly understood andmay vary depending on the pathological context; however, animal datasuggests that disparate atrophic stimuli converge on the activation ofprotein degradation, particularly the ubiquitin (Ub)-26S proteasomalpathway. Two novel Ub-protein ligases, atrogin-1 (muscle atrophy F-boxprotein) and muscle ring finger protein (MuRF-1), are consistentlyup-regulated in murine models of muscle atrophy, and are thought toubiquitinate both regulatory (e.g., calcineurin and MyoD) and structural(e.g., myosin and troponin I) proteins, thus directing the specificdegradation of proteins during muscle atrophy (Abadi et al., PLoS ONE4(8):e6518 (2009)).

While much progress has been made towards delineating the underlyingfunctional alterations and signaling pathways that mediate muscleatrophy in animal models, few studies have examined muscle atrophy inhumans. Early reports concerning protein turnover in humans demonstratedthat mixed muscle protein synthesis rates decline during muscle atrophywhile protein degradation rates appear unchanged (de Grey, Curr. DrugTargets 7:1469-1477 (2006); Ferrando et al., Am. J. Physiol.270:E627-633 (1996); Gibson et al., Clin. Sci. (Lond) 72:503-509 (1987);Shangraw et al., Am. J. Physiol. 255:E548-558 (1988)). This wasconfirmed in a recent study in which the rate of myofibrillar proteinsynthesis decreased by 50% following 10 d of unilateral limb suspension(ULS) in human subjects (de Boer et al., J. Physiol. 585:241-251(2007)). These studies have emphasized the suppression of proteinsynthesis during atrophy in human muscle, which contrasts with studiesin murine models that point primarily towards increased proteindegradation. However, one recent study found that myofibrillar proteindegradation was increased in humans as early as 72 h following ULS(Tesch et al., J. Appl. Physiol. 105:902-906 (2008)). In addition, theexpression of atrogin-1 and MuRF-1 during muscle atrophy in humans iscontentious, with some studies showing increased atrogin-1 and MuRF-1mRNA, but not others (Abadi et al., PLoS ONE 4(8):e6518 (2009)).

In a study conducted by Abadi and colleagues (Abadi et al., PLoS ONE4(8):e6518 (2009)), the transcriptional suppression of bioenergetic andmitochondrial genes dominated the immobilization-induced transcriptionand was evident as early as 48 hours following immobilization. Thesetranscriptional changes were accompanied by declines in both the proteinlevel and enzymatic activity of several mitochondrial proteins following14 days of immobilization. In addition, atrogin-1 and MuRF-1 mRNA wassignificantly up-regulated early during the progression of muscleatrophy, and protein ubiquitination was increased following 48 hours ofimmobilization, but not following 14 days of immobilization. Lastly,mTOR phosphorylation decreased significantly following 48 hours ofimmobilization, but not following 14 days of immobilization.

Existing treatments for muscle atrophy include exercise or physicaltherapy (when possible), functional electrical stimulation of muscles,and amino acid therapy (e.g., administration of branched-chain aminoacids (BAAs)) to attempt to regenerate damaged or atrophied muscletissue. In severe cases of muscle atrophy, anabolic steroids such asmethandrostenolone have been administered to patients. However, theefficacy of existing treatments has been limited, and the use of BAAsand anabolic steroids are both known to produce side effects. Forexample, BAAs can cause fatigue and loss of coordination, while anabolicsteroids can cause cardiovascular disease, impaired liver function, andboth estrogenic and androgenic effects (e.g., acne, body/facial hairgrowth, male pattern baldness, and gynecomastia). Accordingly, thereremains a need for improved therapies for the treatment of muscleatrophy.

The present invention relates to the use of a PPARδ agonist to treatmuscle atrophy in a subject in need thereof.

BRIEF SUMMARY OF THE INVENTION

Certain variations of the present invention provide improved treatmentof muscle atrophy by administering a PPARδ agonist to a subject in needthereof.

The present invention is directed to a method of treatingdisuse-associated muscle atrophy in a subject in need thereof comprisingadministering to the subject an effective amount of a PPARδ agonist. Inone embodiment, the PPARδ agonist is selected from the group consistingof:

-   (Z)-[2-Methyl-4-[3-(4-methylphenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-phenoxy]acetic    acid;-   (E)-[2-Methyl-4-[3-[4-[3-(pyrazol-1-yl)prop-1-ynyl]phenyl]-3-(4-trifluoromethylphenyl)-allyloxy]phenoxy]acetic    acid;-   (E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]acetic    acid;-   (E)-[2-Methyl-4-[3-[4-[3-(morpholin-4-yl)propynyl]phenyl]-3-(4-trifluoromethylphenyl)allyloxy]-phenoxy]acetic    acid;-   (E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]acetic    acid;-   (E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methylphenyl]-propionic    acid;-   {4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-benzylsulfanyl]-2-methyl-phenoxy}-acetic    acid;-   {4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-phenylsulfanyl]-2-methyl-phenoxy}-acetic    acid; and-   {4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-2-methyl-phenoxy}-acetic    acid;    or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the PPARδ agonist is(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention is directed to a method forreducing disuse-associated muscle atrophy in a subject in need thereofcomprising administering to the subject an effective amount of a PPARδagonist. In a particular embodiment, the disuse-associated muscleatrophy is caused by limb immobilization in the subject. In anotherparticular embodiment, the disuse-associated muscle atrophy is caused byuse of a mechanical ventilator by the subject.

In one embodiment, reducing disuse-associated muscle atrophy comprisesreducing the rate of loss of muscle strength in a muscle tissue of thesubject relative to a control, wherein the rate of loss of musclestrength comprises a comparison of one or more measurements of musclestrength in the subject to a baseline measurement of muscle strength inthe same subject prior to a period of disuse, wherein muscle strength ismeasured by a muscle strength test. In another embodiment, reducing therate of loss of muscle strength in the subject comprises a return to thesubject's baseline measurement of muscle strength faster than thecontrol following a period of disuse. In another embodiment, the loss ofmuscle strength in the subject is less than the loss of muscle strengthrelative to the control during a period of disuse.

In another embodiment, reducing disuse-associated muscle atrophycomprises reducing the rate of loss of muscle mass in a muscle tissue ofthe subject relative to a control, wherein the rate of loss of musclemass comprises a comparison of one or more measurements of muscle volumein the subject to a baseline measurement of muscle volume in the samesubject prior to a period of disuse, wherein muscle volume is measuredby the cross-section area of a muscle. In another embodiment, reducingthe rate of loss of muscle mass in the subject comprises a return to thesubject's baseline measurement of muscle mass faster than the controlfollowing a period of disuse. In another embodiment, the loss of musclemass in the subject is less than the loss of muscle mass relative to thecontrol during a period of disuse.

In another embodiment, reducing disuse-associated muscle atrophycomprises reducing the rate of loss of Type I muscle fibers in a muscletissue of the subject relative to a control, wherein the rate of loss ofType I muscle fibers comprises a comparison of one or more measurementsof Type I muscle fibers in the subject to a baseline measurement of TypeI muscle fibers in the same subject. In an embodiment, the amount ofType I muscle fibers is measured by using myosin ATPase staining ofmuscle samples. In another embodiment, reducing the rate of loss of TypeI muscle fibers in the subject comprises a return to the subject'sbaseline measurement of Type I muscle fibers faster than the controlfollowing a period of disuse. In another embodiment, the loss of Type Imuscle fibers in the subject is less than the loss of Type I musclefibers relative to the control during a period of disuse.

In another embodiment, reducing disuse-associated muscle atrophycomprises reducing the rate of decrease in mitochondrial biogenesis in amuscle tissue of the subject relative to a control, wherein the rate ofdecrease in mitochondrial biogenesis comprises a comparison of one ormore measurements of mitochondrial biogenesis in the subject to abaseline measurement of mitochondrial biogenesis in the same subject. Inanother embodiment, reducing the rate of decrease in mitochondrialbiogenesis in the subject comprises a return to the subject's baselinemeasurement of mitochondrial biogenesis faster than the controlfollowing a period of disuse. In another embodiment, the decrease inmitochondrial biogenesis in the subject is less than the decrease inmitochondrial biogenesis relative to the control during a period ofdisuse.

In another embodiment, the methods of the present invention for reducingdisuse-associated muscle atrophy comprise administration of a PPARδagonist to a subject in need thereof before, during, or after a periodof disuse, or any combination thereof.

This Summary is provided merely to introduce certain concepts, and isnot intended to identify any key or essential features of the claimedsubject matter.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a graph of mean changes from baseline in muscle strengthrepresenting the effect of administration of a PPARδ agonist onperformance of a repeated measures knee extension strength test during(day 0 to day 14) and after (day 14 to day 21 and day 21 to day 29) limbimmobilization in human subjects. Data reflects multiple imputation formissing and invalid data.

FIG. 2 shows a graph of mean changes from baseline in muscle strengthrepresenting the effect of administration of a PPARδ agonist onperformance of a repeated measures knee extension strength test during(day 0 to day 14) and after (day 14 to day 21 and day 21 to day 29) limbimmobilization in human subjects. Data reflects all available data forsubjects with valid data, excluding protocol violators (i.e., noimputation for missing and invalid data).

DETAILED DESCRIPTION

As used herein, the PPARδ agonist compounds of the present invention areuseful in treating muscle atrophy in a subject in need thereof.

PPARδ is the most abundant PPAR isoform in skeletal muscle and has ahigher expression in oxidative type I muscle fibers compared withglycolytic type II muscle fibers (Wang et al., PLoS Biol. 2:e294(2004)). Both short-term exercise and endurance training lead toincreased PPARδ expression in human and rodent skeletal muscle (Watt etal., J. Mol. Endocrinol. 33:533-544 (2004); Mahoney et al., FASEB J.19:1498-1500 (2005); Russell et al., Diabetes 52:2874-2881 (2003); andFritz et al., Diabetes Metab. Res. Rev. 2:492-498 (2006)). Rodentstudies suggest that a key feature of PPARδ activation is induction ofskeletal muscle fatty acid oxidation (Tanaka et al., Proc. Natl. Acad.Sci. U.S.A. 100:15924-15929 (2003); Wang et al., Cell 113:159-170(2003)). On activation of PPARδ in skeletal muscle in mice, the fibercomposition changes toward the oxidative type I with induction of fattyacid oxidation, mitochondrial respiration, oxidative metabolism, andslow-twitch contractile apparatus. In addition to the metabolic effects,this study also demonstrated that PPARδ stimulated peroxisomeproliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), aneffect accompanied by mitochondrial biogenesis (Tanaka et al., Proc.Natl. Acad. Sci. U.S.A. 100:15924-15929 (2003)). Interestingly, thistype of adaptation is identical to that seen in response to physicalexercise, and indeed, mice with transgenic (Tg) overexpression of PPARδexhibit increased running endurance (Wang et al., PLoS Biol. 2:e294(2004)).

Methods of Treatment

The present invention is generally directed to methods of treatingmuscle atrophy in a subject in need thereof comprising administering tothe subject an effective amount of a PPARδ agonist.

A muscle is a soft tissue found in most animals comprising muscle cells.Muscle cells contain protein filaments that can slide past one anotherand produce a contraction that changes both the length and shape of themuscle cell. Muscles function to produce force and motion. There arethree types of muscles in the body: a) skeletal muscle (the muscleresponsible for moving extremities and external areas of the bodies); b)cardiac muscle (the heart muscle); and c) smooth muscle (the muscle thatis in the walls of arteries and bowel).

The term “muscle cell” as used herein refers to any cell thatcontributes to muscle tissue. Myoblasts, satellite cells, myotubes, andmyofibril tissues are all included in the term “muscle cells” and mayall be treated using the methods of the invention. Muscle cell effectsmay be induced within skeletal, cardiac, and smooth muscles.

Skeletal muscle, or voluntary muscle, is generally anchored by tendonsto bone and is generally used to effect skeletal movement such aslocomotion or in maintaining posture. Although some control of skeletalmuscle is generally maintained as an unconscious reflex (e.g., posturalmuscles or the diaphragm), skeletal muscles react to conscious control.Smooth muscle, or involuntary muscle, is found within the walls oforgans and structures such as the esophagus, stomach, intestines,uterus, urethra, and blood vessels. Unlike skeletal muscle, smoothmuscle is not under conscious control. Cardiac muscle is also aninvoluntary muscle but more closely resembles skeletal muscle instructure and is found only in the heart. Cardiac and skeletal musclesare striated in that they contain sarcomeres that are packed into highlyregular arrangements of bundles. By contrast, the myofibrils of smoothmuscle cells are not arranged in sarcomeres and therefore are notstriated.

Skeletal muscle is further divided into two broad types: Type I (or“slow twitch”) and Type II (or “fast twitch”). Type I muscle fibers aredense with capillaries and are rich in mitochondria and myoglobin, whichgives Type I muscle tissue a characteristic red color. Type I musclefibers can carry more oxygen and sustain aerobic activity using fats orcarbohydrates for fuel. Type I muscle fibers contract for long periodsof time but with little force. Type II muscle fibers may be subdividedinto three major subtypes (IIa, IIx, and IIb) that vary in bothcontractile speed and force generated. Type II muscle fibers contractquickly and powerfully but fatigue very rapidly, and therefore produceonly short, anaerobic bursts of activity before muscle contractionbecomes painful.

“Muscle atrophy” as used herein refers to a loss of muscle mass and/orto a progressive weakening and degeneration of muscles. The loss ofmuscle mass and/or the progressive weakening and degeneration of musclescan occur because of an unusually high rate of protein degradation, anunusually low rate of protein synthesis, or a combination of both. Anunusually high rate of muscle protein degradation can occur due tomuscle protein catabolism (i.e., the breakdown of muscle protein inorder to use amino acids as substrates for gluconeogenesis).

In another embodiment, muscle atrophy refers to significant loss inmuscle strength. By significant loss in muscle strength is meant areduction of strength in diseased, injured, or unused muscle tissue in asubject relative to the same muscle tissue in a control subject. In anembodiment, a significant loss in muscle strength may be a reduction instrength of at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, ormore relative to the same muscle tissue in a control subject. In anotherembodiment, by significant loss in muscle strength is meant a reductionof strength in unused muscle tissue relative to the muscle strength ofthe same muscle tissue in the same subject prior to a period of nonuse.In an embodiment, a significant loss in muscle strength may be areduction of at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, ormore relative to the muscle strength of the same muscle tissue in thesame subject prior to a period of nonuse. Muscle strength may bemeasured by a muscle strength test (see, e.g., Muscle Strength Testmethods as described in the Examples below).

In another embodiment, muscle atrophy refers to significant loss inmuscle mass. By significant loss in muscle mass is meant a reduction ofmuscle volume in diseased, injured, or unused muscle tissue in a subjectrelative to the same muscle tissue in a control subject. In anembodiment, a significant loss of muscle volume may be at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, or more relative to the samemuscle tissue in a control subject. In another embodiment, bysignificant loss in muscle mass is meant a reduction of muscle volume inunused muscle tissue relative to the muscle volume of the same muscletissue in the same subject prior to a period of nonuse. In anembodiment, a significant loss in muscle tissue may be at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, or more relative to the musclevolume of the same muscle tissue in the same subject prior to a periodof nonuse. Muscle volume may be measured by evaluating the cross-sectionarea of a muscle such as by Magnetic Resonance Imaging (MRI; see, e.g.,muscle volume/cross-section area (CSA) MRI methods as described in theExamples below).

Among the general population, most muscle atrophy results from disuse.Disuse-associated muscle atrophy can result when a limb is immobilized(e.g., due to a limb or joint fracture or an orthopedic surgery such asa hip or knee replacement surgery). As used herein, “immobilization” or“immobilized” refers to the partial or complete restriction of movementof limbs, muscles, bones, tendons, joints, or any other body parts foran extended period of time (e.g., for 2 days, 3 days, 4 days, 5 days, 6days, a week, two weeks, or more). A period of immobilization mayinclude short periods or instances of unrestrained movement, such as tobathe, to replace an external device, or to adjust an external device.Limb immobilization may be carried out by any variety of externaldevices including, but not limited to, braces, slings, casts, bandages,and splints (any of which may be composed of hard or soft materialincluding but not limited to cloth, gauze, fiberglass, plastic, plaster,or metal), as well as any variety of internal devices includingsurgically implanted splints, plates, braces, and the like. In thecontext of limb immobilization, the restriction of movement may involvea single joint or multiple joints (e.g., simple joints such as theshoulder joint or hip joint, compound joints such as the radiocarpaljoint, and complex joints such as the knee joint, including but notlimited to one or more of the following: articulations of the hand,shoulder joints, elbow joints, wrist joints, auxiliary articulations,sternoclavicular joints, vertebral articulations, temporomandibularjoints, sacroiliac joints, hip joints, knee joints, and articulations ofthe foot), a single tendon or ligament or multiple tendons or ligaments(e.g., including but not limited to one or more of the following: theanterior cruciate ligament, the posterior cruciate ligament, rotatorcuff tendons, medial collateral ligaments of the elbow and knee, flexortendons of the hand, lateral ligaments of the ankle, and tendons andligaments of the jaw or temporomandibular joint), a single bone ormultiple bones (e.g., including but not limited to one or more of thefollowing: the skull, mandible, clavicle, ribs, radius, ulna, humerous,pelvis, sacrum, femur, patella, phalanges, carpals, metacarpals,tarsals, metatarsals, fibula, tibia, scapula, and vertabrae), a singlemuscle or multiple muscles (e.g., including but not limited to one ormore of the following: latissimus dorsi, trapezius, deltoid, pectorals,biceps, triceps, external obliques, abdominals, gluteus maximus,hamstrings, quadriceps, gastrocnemius, and diaphragm); a single limb ormultiple limbs (e.g., one or more of the arms and legs), or the entireskeletal muscle system or portions thereof (e.g., in the case of a fullbody cast or spica cast).

Disuse-associated muscle atrophy can also result when the use of a limbis reduced (e.g., due to joint pain associated with rheumatoid arthritisor injury), or due to a prolonged period of inactivity due to illness,bed rest, or a debilitative state.

Disuse-associated muscle atrophy can also result from the use ofmechanical ventilation by a subject. Even though mechanical ventilationis a life-saving measure for subjects with respiratory failure,complications associated with weaning patients from mechanicalventilation are common, in particular due to respiratory muscle weaknessof the diaphragm, a skeletal muscle.

Accordingly, in one embodiment, the present invention is directed to amethod for reducing disuse-associated muscle atrophy in a subject inneed thereof comprising administering to the subject an effective amountof a PPARδ agonist. In a particular embodiment, the disuse-associatedmuscle atrophy is caused by limb immobilization in the subject. Inanother particular embodiment, the disuse-associated muscle atrophy iscaused by use of a mechanical ventilator by the subject.

In one embodiment, reducing disuse-associated muscle atrophy comprisesreducing the rate of loss of muscle strength in a muscle tissue of thesubject relative to a control, wherein the rate of loss of musclestrength comprises a comparison of one or more measurements of musclestrength in the subject to a baseline measurement of muscle strength inthe same subject, wherein muscle strength is measured by a musclestrength test (see, e.g., Muscle Strength Test methods as described inthe Examples below). In another embodiment, reducing the rate of loss ofmuscle strength in the subject comprises a return to the subject'sbaseline measurement of muscle strength faster than the controlfollowing a period of disuse. In a further embodiment, reducing the rateof loss of muscle strength in the subject comprises a return to thesubject's baseline measurement of muscle strength following a period ofdisuse in less than 95%, or less than 90%, or less than 85%, or lessthan 80%, or less than 75%, or less than 70%, or less than 65%, or lessthan 60%, or less than 55%, or less than 50% of the time to return tobaseline for a control. In another embodiment, the loss of musclestrength in the subject is less than the loss of muscle strengthrelative to the control. In a further embodiment, the loss of musclestrength in the subject comprises less than a 50%, less than a 45%, lessthan a 40%, less than a 35%, less than a 30%, less than a 25%, less thana 20%, less than a 15%, less than a 10%, less than a 9%, less than an8%, less than a 7%, less than a 6%, less than a 5%, less than a 4%, lessthan a 3%, less than a 2%, less than a 1%, or a 0% loss of musclestrength relative to the subject's baseline measurement of musclestrength prior to a period of disuse.

In another embodiment, reducing disuse-associated muscle atrophycomprises reducing the rate of loss of muscle mass in a muscle tissue ofthe subject relative to a control, wherein the rate of loss of musclemass comprises a comparison of one or more measurements of muscle volumein the subject to a baseline measurement of muscle volume in the samesubject, wherein muscle volume is measured by the cross-section area ofa muscle (such as by Magnetic Resonance Imaging [MRI]; see, e.g., musclevolume/cross-section area [CSA] MRI methods as described in the Examplesbelow). In another embodiment, reducing the rate of loss of muscle massin the subject comprises a return to the subject's baseline measurementof muscle mass faster than the control. In a further embodiment,reducing the rate of loss of muscle mass in the subject comprises areturn to the subject's baseline measurement of muscle mass following aperiod of disuse in less than 95%, or less than 90%, or less than 85%,or less than 80%, or less than 75%, or less than 70%, or less than 65%,or less than 60%, or less than 55%, or less than 50% of the time toreturn to baseline for a control. In another embodiment, the loss ofmuscle mass in the subject is less than the loss of muscle mass relativeto the control. In a further embodiment, the loss of muscle mass in thesubject comprises less than a 50%, less than a 45%, less than a 40%,less than a 35%, less than a 30%, less than a 25%, less than a 20%, lessthan a 15%, less than a 10%, less than a 9%, less than an 8%, less thana 7%, less than a 6%, less than a 5%, less than a 4%, less than a 3%,less than a 2%, less than a 1%, or a 0% loss of muscle mass relative tothe subject's baseline measurement of muscle mass prior to a period ofdisuse.

In another embodiment, reducing disuse-associated muscle atrophycomprises reducing the rate of loss of Type I muscle fibers in a muscletissue of the subject relative to a control, wherein the rate of loss ofType I muscle fibers comprises a comparison of one or more measurementsof Type I muscle fibers in the subject to a baseline measurement of TypeI muscle fibers in the same subject, wherein Type I muscle fibers ismeasured by using myosin ATPase staining. In another embodiment,reducing the rate of loss of Type I muscle fibers in the subjectcomprises a return to the subject's baseline measurement of Type Imuscle fibers faster than the control. In a further embodiment, reducingthe rate of loss of Type I muscle fibers in the subject comprises areturn to the subject's baseline measurement of Type I muscle fibersfollowing a period of disuse in less than 95%, or less than 90%, or lessthan 85%, or less than 80%, or less than 75%, or less than 70%, or lessthan 65%, or less than 60%, or less than 55%, or less than 50% of thetime to return to baseline for a control. In another embodiment, theloss of Type I muscle fibers in the subject is less than the loss ofType I muscle fibers relative to the control. In a further embodiment,the loss of Type I muscle fibers in the subject comprises less than a50%, less than a 45%, less than a 40%, less than a 35%, less than a 30%,less than a 25%, less than a 20%, less than a 15%, less than a 10%, lessthan a 9%, less than an 8%, less than a 7%, less than a 6%, less than a5%, less than a 4%, less than a 3%, less than a 2%, less than a 1%, or a0% loss of Type I muscle fibers relative to the subject's baselinemeasurement of Type I muscle fibers prior to a period of disuse.

Procedures for measuring Type I muscle fibers are described in N. Yasudaet al. J Appl Physiol 99: 1085-1092 (2005). For example, musclespecimens may be dissected of visible fat and connective tissue andplaced into optimum cutting temperature embedding medium (OCTTissue-Tek) with the orientation of the fibers perpendicular to thehorizontal plane. The samples may be quickly frozen in isopentane,cooled by liquid nitrogen, and stored at −80° C. until subsequenthistochemical analysis. At histochemical analysis, the OCT-mountedmuscle samples may be serially sectioned to 10-μm thickness, and Type I,IIa, and IIx muscle fibers may be determined by using myosin ATPasestaining.

In another embodiment, reducing disuse-associated muscle atrophycomprises reducing the rate of decrease in mitochondrial biogenesis in amuscle tissue of the subject relative to a control, wherein the rate ofdecrease in mitochondrial biogenesis comprises a comparison of one ormore measurements of mitochondrial biogenesis in the subject to abaseline measurement of mitochondrial biogenesis in the same subject. Inanother embodiment, reducing the rate of decrease in mitochondrialbiogenesis in the subject comprises a return to the subject's baselinemeasurement of mitochondrial biogenesis faster than the control. In afurther embodiment, reducing the rate of decrease in mitochondrialbiogenesis in the subject comprises a return to the subject's baselinemeasurement of mitochondrial biogenesis following a period of disuse inless than 95%, or less than 90%, or less than 85%, or less than 80%, orless than 75%, or less than 70%, or less than 65%, or less than 60%, orless than 55%, or less than 50% of the time to return to baseline for acontrol. In another embodiment, the decrease in mitochondrial biogenesisin the subject is less than the decrease in mitochondrial biogenesisrelative to the control. In a further embodiment, the decrease inmitochondrial biogenesis in the subject comprises less than a 50%, lessthan a 45%, less than a 40%, less than a 35%, less than a 30%, less thana 25%, less than a 20%, less than a 15%, less than a 10%, less than a9%, less than an 8%, less than a 7%, less than a 6%, less than a 5%,less than a 4%, less than a 3%, less than a 2%, less than a 1%, or a 0%decrease in mitochondrial biogenesis relative to the subject's baselinemeasurement of mitochondrial biogenesis prior to a period of disuse.

Mitochondrial biogenesis is measured by mitochondrial mass and volumethrough histological section staining using a fluorescently labeledantibody specific to the oxidative-phosphorylation complexes, such asthe Anti-OxPhox Complex Vd subunit antibody from Life Technologies orusing mitochondrial specific dyes in live cell staining, such as theMito-tracker probes from Life Technologies. Mitochondrial biogenesis canalso be measured by monitoring the gene expression of one or moremitochondrial biogenesis related transcription factors such as PGC1a,NRF1, or NRF2 using a technique such as QPCR.

In another embodiment, the method of the invention comprises a methodfor treating muscle atrophy caused by time spent in a zero gravity,reduced gravity, or perceived zero gravity environment in a subject inneed thereof comprising administering to the subject an effective amountof a PPARδ agonist.

Muscle atrophy can also be associated with disease. Disease-associatedmuscle atrophy is less common than disuse-associated muscle atrophy andcan result from diseases that either affect the nerves that supplyindividual muscles (i.e., neurogenic atrophy) or from diseases intrinsicto muscle tissue (i.e., muscle disease). In neurogenic atrophy, thenerve supply to the muscle can be interrupted or compromised bycompression, injury, or disease within the nerve cells, resulting in atemporary or permanent nerve deficit. Diseases within nerve cells thatcan interrupt or compromise nerve supply to muscles include, forexample, multiple sclerosis, amyotrophic lateral sclerosis (ALS, or LouGehrig's disease), Guillain-Barré syndrome, stroke, and viral infectionof nerve cells (e.g., poliomyelitis). Muscle diseases can be intrinsicto muscle tissue (e.g., muscular dystrophy, polymyositis, or myotonia)or can occur as a response to systemic illness (e.g., hypo- orhyperthyroidism, adrenal gland depletion, diabetes mellitus, orautoimmune diseases). Sarcopenia is a debilitating disease that afflictsthe elderly and is characterized by loss of muscle mass and functionwith advanced age. Generalized muscle wasting (cachexia) can also occuras a secondary consequence of such diseases as advanced cancer, AcquiredImmune Deficiency Syndrome (AIDS), chronic obstructive lung disease,congestive heart failure, cardiomyopathy, chronic liver disease, renaldisease, emphysema, tuberculosis, osteomalacia, hormonal deficiency,anorexia nervosa, generalized malnutrition, and drug abuse (e.g., abuseof alcohol, opiates, or steroids).

In another embodiment, the present invention provides methods to inhibitmuscle atrophy and/or to increase muscle mass by providing to a subjectin need thereof an effective amount of PPARδ agonist compound, andpharmaceutical compositions comprising compounds used in the methods. Inanother embodiment, the present invention provides methods to modulatemuscle growth, or to increase muscle strength, or to maintain musclestrength, or to reduce loss of muscle strength, or to induce skeletalmuscle hypertrophy, or to enhance tissue growth in vitro or in vivo, orto enhance muscle formation, and pharmaceutical compositions comprisingcompounds used in these methods. In each of these methods andpharmaceutical compositions, a PPARδ agonist compound is administered orused.

In another embodiment, the present invention provides a kit comprisingat least one PPARδ agonist compound and one or more of: (a) a proteinsupplement; (b) an anabolic agent; (c) a catabolic agent; (d) a dietarysupplement; (e) at least one agent known to treat a disorder associatedwith muscle wasting; (f) instructions for treating a disorder associatedwith cholinergic activity; or (g) instructions for using the compound toincrease muscle mass and/or muscular strength. The kits can alsocomprise compounds and/or products co-packaged, co-formulated, and/orco-delivered with other components. For example, a drug manufacturer, adrug reseller, a physician, a compounding shop, or a pharmacist canprovide a kit comprising a PPARδ agonist compound and/or product andanother component for delivery to a patient. It is contemplated that thedisclosed kits can be used in connection with the disclosed methods ofmaking, the disclosed methods of using, and/or the disclosedcompositions.

In another embodiment, a PPARδ agonist compound may be used in thetreatment of muscle disorders. The muscle disorder can be skeletalmuscle atrophy secondary to malnutrition, muscle disuse (secondary tovoluntary or involuntary bed rest), neurologic disease (includingmultiple sclerosis, amyotrophic lateral sclerosis, spinal muscularatrophy, critical illness neuropathy, spinal cord injury or peripheralnerve injury), orthopedic injury, casting, and other post-surgical formsof limb immobilization, chronic disease (including cancer, congestiveheart failure, chronic pulmonary disease, chronic renal failure, chronicliver disease, diabetes mellitus, Cushing syndrome, and chronicinfections such as HIV/AIDS or tuberculosis), burns, sepsis, otherillnesses requiring mechanical ventilation, drug-induced muscle disease(such as glucorticoid-induced myopathy and statin-induced myopathy),genetic diseases that primarily affect skeletal muscle (such as musculardystrophy and myotonic dystrophy), autoimmune diseases that affectskeletal muscle (such as polymyositis and dermatomyositis), spaceflight,periods of exposure to zero or low gravity, or age-related sarcopenia.Thus, provided is a method for treating or preventing muscle atrophy ina subject suffering from one of these disorders or subject to one ofthese conditions, comprising administering to a subject a PPARδ agonistcompound in an effective amount.

In another embodiment, the present invention provides a method oftreating acute respiratory distress syndrome (ARDS) in a subjectcomprising administering to a subject a PPARδ agonist compound in aneffective amount. In a further embodiment, the subject is on amechanical ventilator. In a further embodiment, the method comprisesreduction in muscle atrophy in the diaphragm.

In another embodiment, the present invention provides a method ofreducing the period to weaning from a mechanical ventilator comprisingadministering to a subject a PPARδ agonist compound in an effectiveamount. In an embodiment, the period to weaning is reduced by at least 1hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 8hours, at least 16 hours, at least 24 hours, at least 32 hours, at least40 hours, at least 48 hours, at least 56 hours, at least 64 hours, or atleast 72 hours.

In another embodiment, the decision to wean from a mechanical ventilatoris evaluated using a manual muscle test (MMT) score. An MMT proximalsubscore (5 muscle groups) may be initially assessed (such as prior toadministration of the PPARδ agonist) and every 3 (±1) days thereafterafter the initial assessment until hospital discharge, including the dayof discharge or the day before. During periods of mechanicalventilation, MMT may be scheduled during sedation holiday. The MMT totalscore (12 muscle groups) may be performed one day after an order hasbeen written for discharge from the ICU and every 7 (±1) days thereafteruntil hospital discharge. The muscle groups that may be assessed arebilateral shoulder abduction, elbow flexion, wrist extension, hipflexion, knee extension, and foot dorsiflexion. In another embodiment,the muscle groups that may be assessed include any grouping of thefollowing: Trapezius (shoulder elevators); Deltoid middle (shoulderabductors); Biceps brachii (elbow flexors); Wrist extensors; Wristflexors; Iliopsoas (hip flexors); Quadriceps femoris (knee extensors);Ankle dorsiflexors; Neck flexors; Gluteus medius (hip abductors); Neckextensors; Gluteus maximus (hip extensors); Hamstrings (knee flexors);and Ankle plantar flexors; including any group of 12.

The subject may be positioned in either the sitting or supine position,depending on the patient's clinical situation. Strength in each musclegroup will be scored according to the six point MRC system, in which ascore of 0 is no contraction; 1 is a flicker of contraction; 2 is activemovement with gravity eliminated; 3 is active movement against gravity;4 is active movement against gravity and resistance; and 5 is normalpower. Proximal muscle strength, an outcome measure, may be scored asthe mean of the scores for bilateral shoulder abduction and bilateralhip flexion, and may be referred to as the MMT proximal subscore.

In another embodiment, the present invention provides a method ofdecreasing the rate of lowering a patient's MMT score (or subscore)wherein the subject is subject to mechanical ventilation, of maintaininga subject's MMT score (or subscore), or increasing a subject's MMT score(or subscore), where the patient is subject to mechanical ventilation,comprising administering to a subject a PPARδ agonist compound in aneffective amount. In an embodiment, the subject's MMT subscore forbilateral shoulder abduction and bilateral hip flexion is 6 or greaterbefore weaning from mechanical ventilation.

In another embodiment, the present invention provides a method ofincreasing the days free of mechanical ventilation for a subject onmechanical ventilation. In an embodiment, the number of days free is outof 28 days. In another embodiment, the present invention provides amethod of increasing the number of hospital free days of a subject onmechanical ventilation. In an embodiment, the number of hospital freedays is out of 28 days.

Also provided is a method for increasing muscle mass, comprisingadministering to a subject a PPARδ agonist compound in an amounteffective to increase the subject's muscle mass. In an embodiment, thesubject is a mammal. In a further embodiment, the mammal is a primate.In a still further embodiment, the mammal is a human. In anotherembodiment, the subject is a domesticated animal. In a furtherembodiment, the domesticated animal is poultry. In an even furtherembodiment, the poultry is selected from chicken, turkey, duck, andgoose. In a still further embodiment, the domesticated animal islivestock. In a further embodiment, the livestock animal is selectedfrom pig, cow, horse, goat, bison, and sheep.

In another embodiment, the present invention provides a method ofenhancing tissue or cell growth in vitro, the method comprisingadministering to the tissue or cells a PPARδ agonist compound in anamount effective to enhance growth of the tissue or cells. In a furtherembodiment, the tissue comprises animal cells. In a still furtherembodiment, the animal cells are muscle cells. In a further embodiment,the muscle cells are myosatellite cells. In an even further embodiment,any of the foregoing tissues or cells may be grown on a scaffold, bead,or other support matrix. In a further embodiment, the present inventionprovides a tissue or cells grown in the presence of a PPARδ agonistcompound. In another embodiment, the tissue or cells grown may beimplanted in a subject from whom the tissue or cells were originallyharvested. In another embodiment, the tissue or cells grown may beimplanted in a subject different from the subject from whom the tissueor cells were originally harvested.

In another embodiment, the present invention provides a method ofenhancing tissue growth in vivo, the method comprising administering aPPARδ agonist compound in an amount effective to enhance growth of atissue or cells following implantation of the tissue or cells into thesubject. In a further embodiment, the tissue comprises animal cells. Ina still further embodiment, the animal cells are muscle cells. In afurther embodiment, the muscle cells are myosatellite cells. In an evenfurther embodiment, any of the foregoing cells may be grown on ascaffold, bead, or other support matrix prior to implantation. In afurther embodiment, the tissue or cells are grown in the presence of aPPARδ agonist compound. In another embodiment, the tissue grown may beimplanted in a subject from whom the tissue or cells were originallyharvested. In another embodiment, the tissue grown may be implanted in asubject different from the subject from whom the tissue or cells wereoriginally harvested.

In another embodiment, the present invention provides uses of a PPARδagonist compound as pharmacological tools in the development andstandardization of in vitro and in vivo test systems for the evaluationof the effects of modulators of muscle hypertrophy or inhibitors ofmuscle atrophy related activity in laboratory animals such as cats,dogs, rabbits, monkeys, rats, and mice, as part of the search for newtherapeutic agents to increase muscle mass and/or inhibit musclehypertrophy.

In any of the embodiments herein where a PPARδ agonist compound isadministered to a subject, the compound may be administeredsystemically, such as by parenteral injection or by oral consumption,and may be used to promote muscle growth and reduce muscle atrophy inall muscles, including those of the limbs and the diaphragm. A PPARδagonist compound may also be administered locally, such as by a topicalroute or localized injection, and may be used to promote local musclegrowth, as can be required following a localized injury or surgery.

In any of the embodiments herein where a PPARδ agonist compound isadministered to a subject, the administration may be combined with aregime of physical therapy to inhibit muscle atrophy, or to increasemuscle mass, or to inhibit loss of muscle strength, or to increasemuscle strength, or to enhance muscle formation.

Accordingly, in an embodiment, the method of the invention comprises amethod for treating a disease associated with muscle atrophy in asubject in need thereof comprising administering to the subject aneffective amount of a PPARδ agonist.

Muscle atrophy can also be associated with injury. Injury-associatedmuscle atrophy can occur, for example, with severe burns and trauma,including, but not limited to, damage to the central nervous system(CNS) or peripheral nervous system (PNS), or exposure to toxicchemicals.

Accordingly, in an embodiment, the method of the invention comprises amethod for treating injury-associated muscle atrophy in a subject inneed thereof comprising administering to the subject an effective amountof a PPARδ agonist.

As used herein, “administer” or “administering” means to introduce, suchas to introduce to a subject a compound(s) or composition. The term isnot limited to any specific mode of delivery, and can include, but isnot limited to, transdermal and oral delivery.

As used herein, “treat” or “treating” or “treatment” can refer to one ormore of: delaying the progress of a disorder; controlling a disorder;delaying the onset of a disorder; ameliorating one or more symptomscharacteristic of a disorder; or delaying the recurrence of a disorder,or characteristic symptoms thereof, depending on the nature of thedisorder and its characteristic symptoms.

In some aspects of the invention, muscle atrophy may be predicted in asubject, for example, in the context of muscle atrophy caused by limbimmobilization or caused by use of a mechanical ventilator by a subject.In such cases, treatment may be initiated prior to the conditionpredicted to cause muscle atrophy. For example, treatment of a subjectwith an effective amount of a PPARδ agonist may be initiated immediatelybefore the condition predicted to cause muscle atrophy (e.g, immediatelybefore limb immobilization or use of a mechanical ventilator). Inanother embodiment, treatment of a subject with an effective amount of aPPARδ agonist may be initiated at least 1 hour, at least 2 hours, atleast 3 hours, at least 4 hours, at least 8 hours, at least 16 hours, atleast 24 hours, at least 32 hours, at least 40 hours, at least 48 hours,at least 56 hours, at least 64 hours, or at least 72 hours before thecondition predicted to cause muscle atrophy (e.g, immediately beforelimb immobilization or use of a mechanical ventilator).

Accordingly, in one embodiment the methods of the present invention forreducing disuse-associated muscle atrophy comprise administration of aPPARδ agonist to a subject in need thereof during a period of disuse. Inanother embodiment, the methods of the present invention for reducingdisuse-associated muscle atrophy comprise administration of a PPARδagonist to a subject in need thereof before a period of disuse. Inanother embodiment, the methods of the present invention for reducingdisuse-associated muscle atrophy comprise administration of a PPARδagonist to a subject in need thereof after a period of disuse. Inanother embodiment, the methods of the present invention for reducingdisuse-associated muscle atrophy comprise administration of a PPARδagonist to a subject in need thereof before, during, or after a periodof disuse, or any combination thereof.

In treating muscle atrophy, diagnosing and assessing the severity of thecondition and/or effectiveness of prevention or treatment is ultimatelyleft to the subject and/or attending physician. However, a number oftools are available for assessing the severity of the condition and/oreffectiveness of prevention or treatment, as described elsewhere herein.

As used herein, “subject” generally refers to a human, but also mayinclude other mammals such as horses, cows, sheep, pigs, mice, rats,dogs, cats, and primates. In an embodiment, the subject is a human. Inanother embodiment, the subject is a mammal who exhibits one or moresymptoms characteristic of a disorder. In another embodiment, thesubject is a human who exhibits one or more symptoms characteristic of adisorder. The term subject does not require one to have any particularstatus or relationship with respect to a hospital, clinic, researchfacility, or physician (e.g., as an admitted patient, a studyparticipant, or the like).

Dosages of the compounds used in the present invention must ultimatelybe set by an attending physician. General outlines of the dosages areprovided herein below. Generally, a suitable dose of a PPARδ agonist, ora pharmaceutically acceptable salt thereof, for administration to ahuman will be in the range of about 0.1 mg/kg per day to about 25 mg/kgper day (e.g., about 0.2 mg/kg per day, about 0.3 mg/kg per day, about0.4 mg/kg per day, about 0.5 mg/kg per day, about 0.6 mg/kg per day,about 0.7 mg/kg per day, about 0.8 mg/kg per day, about 0.9 mg/kg perday, about 1 mg/kg per day, about 2 mg/kg per day, about 3 mg/kg perday, about 4 mg/kg per day, about 5 mg/kg per day, about 6 mg/kg perday, about 7 mg/kg per day, about 8 mg/kg per day, about 9 mg/kg perday, about 10 mg/kg per day, about 15 mg/kg per day, about 20 mg/kg perday, or about 25 mg/kg per day). Alternatively, a suitable dose of aPPARδ agonist, or a pharmaceutically acceptable salt thereof, foradministration to a human will be in the range of from about 0.1 mg/dayto about 1000 mg/day; from about 1 mg/day to about 400 mg/day; or fromabout 1 mg/day to about 300 mg/day. In other embodiments, a suitabledose of a PPARδ agonist, or a pharmaceutically acceptable salt thereof,for administration to a human will be about 1 mg/day, about 2 mg/day,about 3 mg/day, about 4 mg/day, about 5 mg/day, about 6 mg/day, about 7mg/day, about 8 mg/day, about 9 mg/day, about 10 mg/day, about 15mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 55mg/day, about 60 mg/day, about 65 mg/day, about 70 mg/day, about 75mg/day, about 80 mg/day, about 85 mg/day, about 90 mg/day, about 95mg/day, about 100 mg/day, about 125 mg/day, about 150 mg/day, about 175mg/day, about 200 mg/day, about 225 mg/day, about 250 mg/day, about 275mg/day, about 300 mg/day, about 325 mg/day, about 350 mg/day, about 375mg/day, about 400 mg/day, about 425 mg/day, about 450 mg/day, about 475mg/day, or about 500 mg/day. Dosages may be administered more than onetime per day (e.g., two, three, four, or more times per day). In oneembodiment, a suitable dose of a PPARδ agonist, or a pharmaceuticallyacceptable salt thereof, for administration to a human is about 100 mgtwice/day (i.e., a total of about 200 mg/day). In another embodiment, asuitable dose of a PPARδ agonist, or a pharmaceutically acceptable saltthereof, for administration to a human is about 50 mg twice/day (i.e., atotal of about 100 mg/day).

In some aspects of the invention, PPARδ agonist is administered in atherapeutically effective amount to a subject (e.g., a human). As usedherein, the term “effective amount” or “therapeutically effectiveamount” refers to an amount of an active ingredient that elicits thedesired biological or medicinal response, for example, reduction oralleviation of the symptoms of the condition being treated. In someembodiments of the invention, the amount of PPARδ agonist administeredcan vary depending on various factors, including, but not limited to,the weight of the subject, the nature and/or extent of the subject'scondition, etc.

Compounds

A peroxisome proliferator activated receptor—delta (PPARδ) agonist is afatty acid, lipid, protein, peptide, small molecule, or other chemicalentity that binds to the cellular PPARδ and elicits a downstreamresponse, namely gene transcription, either native gene transcription ora reporter construct gene transcription, comparable to endogenousligands such as retinoic acid or comparable to a standard referencePPARδ agonist such as carbacyclin.

In an embodiment, a PPARδ agonist is a selective agonist. As usedherein, a selective PPARδ agonist is viewed as a chemical entity thatbinds to and activates the cellular PPARδ and does not substantiallyactivate the cellular peroxisome proliferator activated receptors—alpha(PPARα) and—gamma (PPARγ). As used herein, a selective PPARδ agonist isa chemical entity that has at least a 10-fold maximum activation (ascompared to endogenous receptor ligand) with a greater than 100-foldpotency for activation of PPARδ relative to either or both of PPARα andPPARγ. In a further embodiment, a selective PPARδ agonist is a chemicalentity that binds to and activates the cellular human PPARδ and does notsubstantially activate either or both of human PPARα and PPARγ. In afurther embodiment, a selective PPARδ agonist is a chemical entity thathas at least a 10 fold, or a 20 fold, or a 30 fold, or a 40 fold, or a50 fold, or a 100 fold potency for activation of PPARδ relative toeither or both of PPARα and PPARγ.

“Activation” here is defined as the abovementioned downstream response,which in the case of PPAR's is gene transcription. Gene transcriptionmay be measured indirectly as downstream production of proteinsreflective of the activation of the particular PPAR subtype under study.Alternatively, an artificial reporter construct may be employed to studythe activation of the individual PPAR's expressed in cells. The ligandbinding domain of the particular receptor to be studied may be fused tothe DNA binding domain of a transcription factor, which producesconvenient laboratory readouts, such as the yeast GAL4 transcriptionfactor DNA binding domain. The fusion protein may be transfected into alaboratory cell line along with a Gal4 enhancer, which effects theexpression of the luciferase protein. When such a system is transfectedinto a laboratory cell line, binding of a receptor agonist to the fusionprotein will result in light emission.

A selective PPARδ agonist may exemplify the above gene transcriptionprofile in cells selectively expressing PPARδ, and not in cellsselectively expressing PPARγ or PPARα. In an embodiment, the cells maybe expressing human PPARδ, PPARγ, and PPARα, respectively.

In a further embodiment, a PPARδ agonist may have an EC50 value of lessthan 5 μm as determined by the PPAR transient transactivation assaydescribed below. In an embodiment, the EC50 value is less than 1 μm. Inanother embodiment, the EC50 value is less than 500 nM. In anotherembodiment, the EC50 value is less than 100 nM. In another embodiment,the EC50 value is less than 50 nM.

The PPAR transient transactivation assay may be based on transienttransfection into human HEK293 cells of two plasmids encoding a chimerictest protein and a reporter protein respectively. The chimeric testprotein may be a fusion of the DNA binding domain (DBD) from the yeastGAL4 transcription factor to the ligand binding domain (LBD) of thehuman PPAR proteins. The PPAR-LBD moiety harbored in addition to theligand binding pocket also has the native activation domain, allowingthe fusion protein to function as a PPAR ligand dependent transcriptionfactor. The GAL4 DBD will direct the chimeric protein to bind only toGal4 enhancers (of which none existed in HEK293 cells). The reporterplasmid contained a Gal4 enhancer driving the expression of the fireflyluciferase protein. After transfection, HEK293 cells expressed theGAL4-DBD-PPAR-LBD fusion protein. The fusion protein will in turn bindto the Gal4 enhancer controlling the luciferase expression, and donothing in the absence of ligand. Upon addition to the cells of a PPARligand, luciferase protein will be produced in amounts corresponding tothe activation of the PPAR protein. The amount of luciferase protein ismeasured by light emission after addition of the appropriate substrate.

Cell Culture and Transfection:

HEK293 cells may be grown in DMEM+10% FCS. Cells may be seeded in96-well plates the day before transfection to give a confluency of50-80% at transfection. A total of 0.8 mg DNA containing 0.64 mgpM1a/gLBD, 0.1 mg pCMVbGal, 0.08 mg pGL2(Gal4)₅, and 0.02 mg pADVANTAGEmay be transfected per well using FuGene transfection reagent accordingto the manufacturer's instructions. Cells may be allowed to expressprotein for 48 h followed by addition of compound.

Plasmids:

Human PPARδ may be obtained by PCR amplification using cDNA synthesizedby reverse transcription of mRNA from human liver, adipose tissue, andplancenta, respectively. Amplified cDNAs may be cloned into pCR2.1 andsequenced. The ligand binding domain (LBD) of each PPAR isoform may begenerated by PCR (PPARδ: aa 128—C-terminus) and fused to the DNA bindingdomain (DBD) of the yeast transcription factor GAL4 by subcloningfragments in frame into the vector pM1 (Sadowski et al. (1992), Gene118, 137), generating the plasmids pM1αLBD, pM1γLBD, and pM1δ. Ensuingfusions may be verified by sequencing. The reporter may be constructedby inserting an oligonucleotide encoding five repeats of the GAL4recognition sequence (Webster et al. (1988), Nucleic Acids Res. 16,8192) into the vector pGL2 promotor (Promega), generating the plasmidpGL2(GAL4)₅. pCMVbGal may be purchased from Clontech and pADVANTAGE maybe purchased from Promega.

Compounds:

Compounds may be dissolved in DMSO and diluted 1:1000 upon addition tothe cells. Compounds may be tested in quadruple in concentrationsranging from 0.001 to 300 μM. Cells may be treated with compound for 24h followed by luciferase assay. Each compound may be tested in at leasttwo separate experiments.

Luciferase Assay:

Medium including test compound may be aspirated and 100 μl PBS including1 mM Mg⁺⁺ and Ca⁺⁺ may be added to each well. The luciferase assay maybe performed using the LucLite kit according to the manufacturer'sinstructions (Packard Instruments). Light emission may be quantified bycounting on a Packard LumiCounter. To measure β-galactosidase activity,25 ml supernatant from each transfection lysate may be transferred to anew microplate. β-Galactosidase assays may be performed in the microwellplates using a kit from Promega and read in a Labsystems AscentMultiscan reader. The β-galactosidase data may be used to normalize(transfection efficiency, cell growth, etc.) the luciferase data.

Statistical Methods:

The activity of a compound may be calculated as fold induction comparedto an untreated sample. For each compound, the efficacy (maximalactivity) may be given as a relative activity compared to Wy14,643 forPPARα, rosiglitazone for PPARγ, and carbacyclin for PPARδ. The EC50 isthe concentration giving 50% of maximal observed activity. EC50 valuesmay be calculated via non-linear regression using GraphPad PRISM 3.02(GraphPad Software, San Diego, Calif.).

In a further embodiment, a PPARδ agonist has a molecular weight of lessthan 1000 g/mol, or a molecular weight of less than 950 g/mol, or amolecular weight of less than 900 g/mol, or a molecular weight of lessthan 850 g/mol, or a molecular weight of less than 800 g/mol, or amolecular weight of less than 750 g/mol, or a molecular weight of lessthan 700 g/mol, or a molecular weight of less than 650 g/mol, or amolecular weight of less than 600 g/mol, or a molecular weight of lessthan 550 g/mol, or a molecular weight of less than 500 g/mol, or amolecular weight of less than 450 g/mol, or a molecular weight of lessthan 400 g/mol, or a molecular weight of less than 350 g/mol, or amolecular weight of less than 300 g/mol, or a molecular weight of lessthan 250 g/mol. In another embodiment, a PPARδ agonist has a molecularweight of greater than 200 g/mol, or a molecular weight of greater than250 g/mol, or a molecular weight of greater than 250 g/mol, or amolecular weight of greater than 300 g/mol, or a molecular weight ofgreater than 350 g/mol, or a molecular weight of greater than 400 g/mol,or a molecular weight of greater than 450 g/mol, or a molecular weightof greater than 500 g/mol, or a molecular weight of greater than 550g/mol, or a molecular weight of greater than 600 g/mol, or a molecularweight of greater than 650 g/mol, or a molecular weight of greater than700 g/mol, or a molecular weight of greater than 750 g/mol, or amolecular weight of greater than 800 g/mol, or a molecular weight ofgreater than 850 g/mol, or a molecular weight of greater than 900 g/mol,or a molecular weight of greater than 950 g/mol, or a molecular weightof greater than 1000 g/mol. Any of the upper and lower limits describedabove in this paragraph may be combined.

In an embodiment, a PPARδ agonist may be a PPARδ agonist compound asdisclosed in any of the following published patent applications: WO97/027847, WO 97/027857, WO 97/028115, WO 97/028137, WO 97/028149, WO98/027974, WO 99/004815, WO 2001/000603, WO 2001/025181, WO 2001/025226,WO 2001/034200, WO 2001/060807, WO 2001/079197, WO 2002/014291, WO2002/028434, WO 2002/046154, WO 2002/050048, WO 2002/059098, WO2002/062774, WO 2002/070011, WO 2002/076957, WO 2003/016291, WO2003/024395, WO 2003/033493, WO 2003/035603, WO 2003/072100, WO2003/074050, WO 2003/074051, WO 2003/074052, WO 2003/074495, WO2003/084916, WO 2003/097607, WO 2004/000315, WO 2004/000762, WO2004/005253, WO 2004/037776, WO 2004/060871, WO 2004/063165, WO2004/063166, WO 2004/073606, WO 2004/080943, WO 2004/080947, WO2004/092117, WO 2004/092130, WO 2004/093879, WO 2005/060958, WO2005/097098, WO 2005/097762, WO 2005/097763, WO 2005/115383, WO2006/055187, WO 2007/003581, and WO 2007/071766.

In another embodiment, a PPARδ agonist may be a compound selected fromthe group consisting of sodelglitazar; lobeglitazone; netoglitazone; andisaglitazone;

-   2-[2-methyl-4-[[3-methyl-4-[[4-(trifluoromethyl)phenyl]methoxy]phenyl]thio]phenoxy]-acetic    acid (See WO 2003/024395);-   (S)-4-[cis-2,6-dimethyl-4-(4-trifluoromethoxy-phenyl)piperazine-1-sulfonyl]-indan-2-carboxylic    acid or a tosylate salt thereof (KD-3010);-   4-butoxy-a-ethyl-3-[[[2-fluoro-4-(trifluoromethyl)benzoyl]amino]methyl]-benzenepropanoic    acid (TIPP-204);-   2-[2-methyl-4-[[[4-methyl-2-[4-(trifluoromethyl)phenyl]-5-thiazolyl]methyl]thio]phenoxy]-acetic    acid (GW-501516);-   2-[2,6    dimethyl-4-[3-[4-(methylthio)phenyl]-3-oxo-1(E)-propenyl]phenoxyl]-2-methylpropanoic    acid (GFT-505); and-   {2-methyl-4-[5-methyl-2-(4-trifluoromethyl-phenyl)-2H-[1,2,3]triazol-4-ylmethylsylfanyl]-phenoxy}-acetic    acid.

In an embodiment, a PPARδ agonist is(Z)-[2-Methyl-4-[3-(4-methylphenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-phenoxy]aceticacid:

An example of the chemical synthesis of(Z)-[2-Methyl-4-[3-(4-methylphenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-phenoxy]aceticacid may be found in Example 3 of PCT Application Pub. No. WO2007/071766.

In an embodiment, a PPARδ agonist is(E)-[2-Methyl-4-[3-[4-[3-(pyrazol-1-yl)prop-1-ynyl]phenyl]-3-(4-trifluoromethylphenyl)-allyloxy]phenoxy]aceticacid:

An example of the chemical synthesis of(E)-[2-Methyl-4-[3-[4-[3-(pyrazol-1-yl)prop-1-ynyl]phenyl]-3-(4-trifluoromethylphenyl)-allyloxy]phenoxy]aceticacid may be found in Example 4 of PCT Application Pub. No. WO2007/071766.

In an embodiment, a PPARδ agonist is(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid:

An example of the chemical synthesis of(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid may be found in Example 10 of PCT Application Pub. No. WO2007/071766.

In an embodiment, a PPARδ agonist is(E)-[2-Methyl-4-[3-[4-[3-(morpholin-4-yl)propynyl]phenyl]-3-(4-trifluoromethylphenyl)allyloxy]-phenoxy]aceticacid:

An example of the chemical synthesis of(E)-[2-Methyl-4-[3-[4-[3-(morpholin-4-yl)propynyl]phenyl]-3-(4-trifluoromethylphenyl)allyloxy]-phenoxy]aceticacid may be found in Example 20 of PCT Application Pub. No. WO2007/071766.

In an embodiment, a PPARδ agonist is(E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid:

An example of the chemical synthesis of(E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid may be found in Example 46 of PCT Application Pub. No. WO2007/071766.

In an embodiment, a PPARδ agonist is(E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methylphenyl]-propionicacid:

An example of the chemical synthesis of(E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methylphenyl]-propionicacid may be found in Example 63 of PCT Application Pub. No. WO2007/071766.

In an embodiment, a PPARδ agonist is{4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-benzylsulfanyl]-2-methyl-phenoxy}-aceticacid:

An example of the chemical synthesis of{4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-benzylsulfanyl]-2-methyl-phenoxy}-aceticacid may be found in Example 9 of PCT Application Pub. No. WO2007/003581.

In an embodiment, a PPARδ agonist is{4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-phenylsulfanyl]-2-methyl-phenoxy}-aceticacid:

An example of the chemical synthesis of{4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-phenylsulfanyl]-2-methyl-phenoxy}-aceticacid may be found in Example 35 of PCT Application Pub. No. WO2007/003581.

In an embodiment, a PPARδ agonist is{4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-2-methyl-phenoxy}-acetic acid:

An example of the chemical synthesis of{4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-2-methyl-phenoxy}-acetic acid maybe found in Example 10 of PCT Application Pub. No. WO 2004/037776.

Accordingly, in an embodiment, a PPARδ agonist may be a compoundselected from the group consisting of:

-   (Z)-[2-Methyl-4-[3-(4-methylphenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-phenoxy]acetic    acid;-   (E)-[2-Methyl-4-[3-[4-[3-(pyrazol-1-yl)prop-1-ynyl]phenyl]-3-(4-trifluoromethylphenyl)-allyloxy]phenoxy]acetic    acid;-   (E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]acetic    acid;-   (E)-[2-Methyl-4-[3-[4-[3-(morpholin-4-yl)propynyl]phenyl]-3-(4-trifluoromethylphenyl)allyloxy]-phenoxy]acetic    acid;-   (E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]acetic    acid;-   (E)-[4-[3-(4-Chlorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methylphenyl]-propionic    acid;-   {4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-benzylsulfanyl]-2-methyl-phenoxy}-acetic    acid;-   {4-[3-Isobutoxy-5-(3-morpholin-4-yl-prop-1-ynyl)-phenylsulfanyl]-2-methyl-phenoxy}-acetic    acid; and-   {4-[3,3-Bis-(4-bromo-phenyl)-allyloxy]-2-methyl-phenoxy}-acetic    acid; or    a pharmaceutically acceptable salt thereof.

In a further embodiment, a PPARδ agonist is(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof.

As used throughout this specification, the term “pharmaceuticallyacceptable salt” refers to salts of a free acid or a free base that arenot biologically undesirable and are generally prepared by reacting thefree base with a suitable organic or inorganic acid or by reacting theacid with a suitable organic or inorganic base. The term may be used inreference to any compound of the present invention. Representative saltsinclude the following salts: Acetate, Benzenesulfonate, Benzoate,Bicarbonate, Bisulfate, Bitartrate, Borate, Bromide, Calcium Edetate,Camsylate, Carbonate, Chloride, Clavulanate, Citrate, Dihydrochloride,Edetate, Edisylate, Estolate, Esylate, Fumarate, Gluceptate, Gluconate,Glutamate, Glycollylarsanilate, Hexylresorcinate, Hydrabamine,Hydrobromide, Hydrochloride, Hydroxynaphthoate, Iodide, Isethionate,Lactate, Lactobionate, Laurate, Malate, Maleate, Mandelate, Mesylate,Methylbromide, Methylnitrate, Methylsulfate, Monopotassium Maleate,Mucate, Napsylate, Nitrate, N-methylglucamine, Oxalate, Pamoate(Embonate), Palmitate, Pantothenate, Phosphate/diphosphate,Polygalacturonate, Potassium, Salicylate, Sodium, Stearate, Subacetate,Succinate, Tannate, Tartrate, Teoclate, Tosylate, Triethiodide,Trimethylammonium, and Valerate. When an acidic substituent is present,such as —COOH, there can be formed the ammonium, morpholinium, sodium,potassium, barium, calcium salt, and the like for use as the dosageform. When a basic group is present, such as amino, or a basicheteroaryl radical, such as pyridyl, there can be formed an acidic salt,such as hydrochloride, hydrobromide, phosphate, sulfate,trifluoroacetate, trichloroacetate, acetate, oxalate, maleate, pyruvate,malonate, succinate, citrate, tartarate, fumarate, mandelate, benzoate,cinnamate, methanesulfonate, ethanesulfonate, picrate, and the like, andinclude acids related to the pharmaceutically acceptable salts listed inStephen M. Berge, et al., Journal of Pharmaceutical Sciences, Vol.66(1), pp. 1-19 (1977).

Pharmaceutical Compositions

In some embodiments of the invention, a PPARδ agonist may be includedwithin a pharmaceutical composition. As used herein, the term“pharmaceutical composition” refers to a liquid or solid composition,preferably solid (e.g., a granulated powder), that contains apharmaceutically active ingredient (e.g., a PPARδ agonist) and at leasta carrier, where none of the ingredients is generally biologicallyundesirable at the administered quantities.

Pharmaceutical compositions incorporating a PPARδ agonist may take anyphysical form that is pharmaceutically acceptable. Pharmaceuticalcompositions for oral administration are particularly preferred. In oneembodiment of such pharmaceutical compositions, an effective amount of aPPARδ agonist is incorporated.

The inert ingredients and manner of formulation of the pharmaceuticalcompositions of the invention are conventional. Known methods offormulation used in pharmaceutical science may be followed. All of theusual types of compositions are contemplated, including, but not limitedto, tablets, chewable tablets, capsules, and solutions. The amount ofthe PPARδ agonist, however, is best defined as the effective amount,that is, the amount of the PPARδ agonist that provides the desired doseto the subject in need of such treatment. The activity of the PPARδagonists does not depend on the nature of the composition, so thecompositions may be chosen and formulated solely for convenience andeconomy. Any of the PPARδ agonists as described herein may be formulatedin any desired form of composition.

Capsules may be prepared by mixing the PPARδ agonist with a suitablediluent and filling the proper amount of the mixture in capsules. Theusual diluents include inert powdered substances such as starch of manydifferent kinds, powdered cellulose, especially crystalline andmicrocrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders.

Tablets may be prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants, and disintegrators, as well as the PPARδ agonist.Typical diluents include, for example, various types of starch, lactose,mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such assodium chloride, and powdered sugar. Powdered cellulose derivatives arealso useful. Typical tablet binders are substances such as starch,gelatin, and sugars such as lactose, fructose, glucose, and the like.Natural and synthetic gums are also convenient, including acacia,alginates, methylcellulose, polyvinylpyrrolidine, and the like.Polyethylene glycol, ethylcellulose, and waxes can also serve asbinders.

A lubricant in a tablet formulation may help prevent the tablet andpunches from sticking in the die. A lubricant can be chosen from suchsolids as talc, magnesium and calcium stearate, stearic acid, andhydrogenated vegetable oils.

Tablet disintegrators are substances that swell when wetted to break upthe tablet and release the compound. They include starches, clays,celluloses, aligns, and gums. More particularly, corn and potatostarches, methylcellulose, agar, bentonite, wood cellulose, powderednatural sponge, cation-exchange resins, alginic acid, guar gum, citruspulp, and carboxymethylcellulose, for example, may be used, as well assodium lauryl sulfate.

Enteric formulations are often used to protect an active ingredient fromthe strongly acidic contents of the stomach. Such formulations arecreated by coating a solid dosage form with a film of a polymer that isinsoluble in acid environments, and soluble in basic environments.Exemplary films are cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropylmethylcellulose acetate succinate.

Tablets are often coated with sugar as a flavor and sealant. The PPARδagonists may also be formulated as chewable tablets by using largeamounts of pleasant-tasting substances such as mannitol in theformulation, as is now well-established practice.

Transdermal patches may be used. Typically, a patch comprises a resinouscomposition in which the active compound(s) will dissolve, or partiallydissolve, and is held in contact with the skin by a film that protectsthe composition. Other, more complicated patch compositions are also inuse, particularly those having a membrane pierced with innumerable poresthrough which the drugs are pumped by osmotic action.

In any embodiment where a PPARδ agonist is included in a pharmaceuticalcomposition, such pharmaceutical compositions may be in a form suitablefor oral use, for example, as tablets, troches, lozenges, aqueous oroily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, or syrups or elixirs. Compositions intended for oral usemay be prepared according to any known method, and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents, and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets may contain the active ingredient in admixturewith non-toxic pharmaceutically acceptable excipients that are suitablefor the manufacture of tablets. These excipients may be for example,inert diluents, such as calcium carbonate, sodium carbonate, lactose,calcium phosphate, or sodium phosphate; granulating and disintegratingagents, for example, corn starch or alginic acid; binding agents, forexample, starch, gelatin, or acacia; and lubricating agents, forexample, magnesium stearate, stearic acid, or talc. The tablets may beuncoated or they may be coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed.

Protocol

A protocol for the experimental evaluation of the impact of a PPARδagonist on muscle atrophy during and following the end of limbimmobilization is provided below. In the protocol, reference to Compound1 refers to(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid sodium salt.

A randomized, investigator and subject blind, sponsor open,placebo-controlled study evaluating the possible impact of Compound 1 onthe recovery of muscle atrophy from limb immobilization is described.Subjects will be leg immobilized using a knee brace (30 degrees flexionon the left leg to allow driving) and will be provided with walkingcrutches such that there is no weight bearing on the immobilized leg.Subjects will be enrolled and randomized to receive Compound 1 orplacebo (both referenced as Study Drug in this protocol). The studyconsists of five periods termed SCR (screening period, Day −35 to −3),BL (baseline period: Day −1 to Day 1 [am]), IMM (limb immobilization andtreatment with study drug, Day 1 [pm] to Day 14), TRE (treatment withstudy drug without limb immobilization, Day 15 to Day 29) and REC (notreatment recovery period, Day 29 to Day 42). There will be six testingdays during the study: Day 1 (baseline), Day 14 (brace off day), Day 16(48 hours after brace off), Day 21, Day 29 (last dose) and Day 42 (finalstudy visit). On each testing day, subjects will undergo the combinationof several testing procedures in sequential order as listed: 1) Morningactivity plus approximately 500-step walk (when domiciled at ClinicalResearch Unit [CRU]); 2) Blood sampling; 3) Standardized breakfast(Study Drug administration will be immediately before the breakfastexcept for Day 42); 4) Muscle biopsy; 5) Leg muscle strength tests (MST)and modified physical performance test (PPT); and 6) Magnetic resonanceimaging (MRI) of the thigh to evaluate thigh muscle volume/cross-sectionarea (CSA).

Baseline testing (Day 1 [am]) and testing on day 14 and 16 will beperformed after subjects have been admitted to the Clinical ResearchUnit (CRU). On Day −1 (before baseline testing), subjects will beadmitted to the CRU in the evening after having been instructed toabstain from exercise, to ingest a standard weight-maintaining diet, andto avoid caffeine and alcohol for three days before being admitted tothe CRU. At 1900 h on Day −1, they will consume a standardized meal andthen fast (except for water) and rest in bed until the next morning. At0730 h on Day 1, they will be asked to use the bathroom, shower, brushtheir teeth, and walk (approximately 500 steps). At 0800 h, subjectswill undergo the testing procedures (including breakfast) describedabove. After completion of all testing procedures, subjects will receivelunch and will then be fitted with the knee brace. Subjects will stay atthe CRU until evening Study Drug dosing time. The first dose of thestudy drug will be administered by the site staff on site around 1900 hand dinner will be provided immediately after dosing. Subjects will bedischarged from the CRU with study drug supply and instructions for athome self-administration. Subjects will be encouraged to walk between4,000 and 6,000 steps per day for the duration of the study (Day 1-Day42) unless otherwise specified.

An outpatient visit at Day 6 (±1 day) will be scheduled for safety labsand PK trough level. Subject should be fasted overnight before the visitand the Study Drug will be administered at the study site immediatelybefore breakfast is served.

On Day 13 (−1 day window), (one day before the end of limbimmobilization), subjects will be admitted to the CRU in the eveningafter having been instructed to ingest a standard weight-maintainingdiet and to avoid caffeine and alcohol for three days before beingadmitted to the CRU. At 1900 h, they will consume a standardized mealand then fast (except for water) and rest in bed until the next morning(Day 14). At 0700 h on Day 14, the knee brace will be removed andsubjects will be asked to use the bathroom, shower, brush their teeth,and walk (approximately 500 steps). At 0800 h, subjects will take thestudy drug under the supervision of site staff and undergo the testingprocedures (including breakfast) described above. After completion ofall testing procedures subjects will return to the CRU, where they willreceive lunch, a snack later in the afternoon, and a standardized dinneras described above at 1900 h. They will also be encouraged to take shortwalks to total 2000 steps (±250 steps) on day 14. On day 15, they willreceive standardized meals the same as in Day 14 and will rest in achair in the CRU except for 4 brief periods of walking approximately 500steps each (2000 steps total±250 steps for entire day). On day 16(testing day), subjects will be asked to use the bathroom, shower, brushtheir teeth, and walk (approximately 500 steps) at 0730 h. At 0800 h,they will undergo the testing procedures (including breakfast) describedabove. After completion of all testing procedures, subjects will receivelunch and will be discharged from the CRU.

On days 21, 29, and 42 (±1 day), subjects will be tested as outpatientsin the CRU after having been instructed to consume a weight maintainingdiet and no caffeine for at least 3 days before the study visit. Theywill arrive in the CRU before 0800 h after an overnight fast, bloodsample should be collected around 0800 h, and the study drug should beadministered right after. The standardized breakfast will be providedimmediately after dosing (except for Day 42) and then testing undergoneas described previously.

Two treatment groups in this study:

-   -   a) Treatment group A: Treated with 100 mg of Compound 1 twice        daily from Day 1 to Day 29.    -   b) Treatment group B: Treated with placebo twice daily from Day        1 to Day 29.

If there is a weight gain at the end of the study (Day 42), a weightloss program will be provided as an option to all subjects.

Diagnosis and Main Criteria for Inclusion:

Key inclusion criteria: 1) Healthy males aged 30 to 55 years, inclusive,at the time of screening; 2) Subjects must be in good health, asdetermined by medical history, physical examination, vital signs,electrocardiogram (ECG), and clinical test results; 3) Not restricted toa wheel-chair or confined to a bed; 4) Weight≥50.0 kg; and 5) BMIbetween 18 and 30.0 kg/m², inclusive, at the time of screening. Keyexclusion criteria: 1) Fasting glucose>110 mg/dL (Screening Visit only);2) Serum creatinine>1.5 mg/dL (Screening Visit and Baseline; if serumcreatinine is >1.5 mg/dL and creatinine clearance is >60 mL/min, thesubject need not be excluded); 3) Troponin I level above the upper limitof normal (ULN; Screening Visit and Baseline); 4) Liver function tests(LFTs)>1.5×ULN (Screening Visit and Baseline); 5) Evidence ofsignificant organ system dysfunction (e.g., diabetes, cardiovasculardisease, cirrhosis, hypogonadism, hypo- or hyperthyroidism;hypertension); 6) Any fluctuations in weight (no more than±2% of bodyweight) by subject self-report in the 3 months prior to the ScreeningVisit; 7) Had received Compound 1 in a previous clinical trial; 8)Smoking within 6 month prior to Day −1; and 9) Michigan AlcoholScreening Test score greater than 2.

Safety Criteria:

Adverse events (AEs), clinical laboratory tests, vital signs, 12-leadelectrocardiogram (ECG), and physical examinations.

All safety analyses will be based on the safety population, comprisingall subjects who are randomized to a treatment group and subsequentlyreceive study medication. Safety variables will be summarized usingdescriptive statistics (mean, standard deviation, median, range, andnumber of observations).

Pharmacokinetics (PK):

Trough PK of Compound 1 at 100 mg BID (twice daily) during 28 days oftreatment. Blood samples for assessment of Compound 1 plasma troughconcentrations will be collected throughout the study. Pre-dose (t=0)blood draws for PK samples were taken within 10 minutes prior to dosingon Day 6, 14, 16, 21, and 29. Plasma samples collected from subjectsreceiving Compound 1 were analyzed for Compound 1 concentrations using apreviously developed and validated bioanalytical method.

All PK analyses will be based on the PK population, comprising allsubjects who received Compound 1. All derived PK parameters, and plasmaCompound 1 concentrations at each scheduled assessment time point, willbe summarized with descriptive statistics (arithmetic and geometricmean, standard deviation, coefficient of variation, median, range, andnumber of observations). Graphical displays of individual subject andmean plasma Compound 1 concentrations across time will also begenerated.

Pharmacodynamics (PD):

PD parameters will be assessed at baseline (Day 1 [am]), Day 14, Day 16,Day 21, Day 29, and Day 42 to measure the changes from baseline to Day14, from Day 14 to Day 16, from Day 14 to Day 21, from Day 14 to Day 29,and from Day 14 to Day 42. PD parameters will be: 1) Muscle StrengthTest (MST); 2) Physical Performance Test (PPT); 3) muscle cross sectionarea (CSA) measurement (via Magnetic Resonance Imaging [MRI]); and 4)muscle tissue biomarker measurement (muscle biopsy). Biomarkersevaluated from muscle tissue were: 1) Gene Expression Analysis (GlobalGene Array); 2) PCG-1α downstream gene profile; 3) Micro RNA; 4) ProteinContent (phospho-mTOR, mTOR, Ub, CS, COX subunit II, COX subunit IV); 5)Enzyme Analysis (Citrate Synthase, COX); and 6) Muscle fiber size. A CSAMRI will not be performed on Day 16, and biomarkers from muscle tissuewill not be evaluated on Day 42.

PD sample collection time will be subject to the available schedule ofeach procedure on each of the test days. Blood samples will be collectedwithin 1 hour of dosing (t=0). Breakfast will be provided at 0800 h (±1hour) immediately after dosing. Muscle biopsy (not performed on Day 42)will be performed at 1000 h (2 hours±15 minutes reference to breakfasttime). Muscle strength and physical performance testing will beperformed after muscle biopsy. Magnetic resonance imaging (MRI) of thethigh will be performed last (not performed on Day 16).

After an overnight fast, approximately 30 ml of venous (antecubital)blood will be collected within 1 hour of dosing time (0800 h), tomeasure safety labs and the following PD parameters: glucose, insulin,hsCRP, Lipid panel (HDL-c, LDL-c, Total Cholesterol, and Triglycerides).Blood samples for the determination of glucose concentration will becollected in chilled tubes containing heparin and analyzed immediatelyafter collection.

A punch biopsy from the quadriceps femoris (˜100 mg) will be obtainedthrough a small cutaneous incision during local anesthesia (lidocain,2%). An aliquot of the muscle tissue will be embedded in TissueTek® forhistology; the remaining muscle tissue will be immediately rinsed inice-cold homogenization buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mMEGTA, 10 mM glycerophosphate, 50 mM NaF, 0.1% Triton-X, 0.1%2-mercaptoethanol, 1 complete protease/phosphatase inhibitor tablet[Roche Diagnostics Ltd, Burgess Hill, UK]) or buffered saline, cleanedoff connective tissue and blood, split into two aliquots (one aliquotshould be around 40 mg) and submerged in liquid nitrogen and then storedat −80° C. until further processing.

The Muscle Strength Test (MST):

The maximal amount of weight that the participant is able to lift forone repetition (1-RM) will be measured on a Hoist multi-station weightmachine for the following exercises: leg press, knee extension, kneeflexion, and bench press. Isokinetic (Cybex) testing of kneeextension/flexion will be done to assess deficiencies in rapid strengthrecruitment. Subjects will be seated on the testing device and strappedin to prevent the pelvis from sliding forward. The movement arm will beadjusted to the subject's leg length and the weight of the leg will bedetermined. Isokinetic testing of the knee extensors and flexors will beperformed at 0°/s, 60°/s and 180°/s. Four to five repetitions at eachmode will be performed with the highest two values used for dataanalysis. Subjects will be familiarized with these procedures during thescreening visit.

The Physical Performance Test (PPT):

To objectively evaluate physical performance, we will administer themodified physical performance test (PPT). The modified PPT is aperformance-based global measure of physical performance that evaluatesthe ability to perform usual daily activities, including both basicactivities of daily living and instrumental activities of daily living.It includes 6 tasks that are timed: 1) climb a flight of 10 stairs, 2)stand up 5 times from a 16″ high chair, 3) walk 50 ft, 4) put on andremove a coat, 5) pick up a penny placed 12″ in front of the foot on thedominant side, and 6) lift a 7 lb book to a shelf ˜12 in above shoulderheight. The other 3 tasks include an evaluation of 1) the ability toclimb up and down 4 flights of 10 stairs, 2) the performance of a 360°turn, and 3) standing balance with feet side-by-side, semi-tandem, andfull-tandem.

Magnetic Resonance Imaging:

MRI will be used to quantify thigh muscle volume. Images will beacquired on a 1.5-T superconducting Siemens MRI scanner (Siemens,Iselin, N.J.) in the Human Imaging Unit facilities at WashingtonUniversity School of Medicine. Bilateral T1-weighted axial images withand without fat saturation will be acquired using commercially availableSiemens sequences starting 10 cm proximal to the distal edge of thefemur and covering an approximate extent of 10 cm. Aftercorrecting/subtracting intramuscular fat, muscle volumes in each of theimages will be determined by segmenting the cross-sectional muscle areasfor each slice using Matlab software (Mathworks, Natick, Mass.) andsumming the area by slice thickness for all slices. The analysis methodwill include a series of semi-automated steps such as imagefiltering/homogeneity correction, tissue identification by thresholdanalysis, manual review/correction of resulting classifications, andreporting of muscle volumes.

PD analysis will be based on the evaluable population. PD variables willbe summarized with descriptive statistics (mean, standard deviation,median, range, and number of observations). Appropriate inferentialanalyses may be performed to evaluate treatment trends on change frombaseline or between-group differences. In particular, there will be amatched pair analysis of each subject in the study. The analysis willcompare PD variable levels prior to drug exposure with tests of subjectplasma during each day of dosing and final study visit. Within-groupchange from baseline to Day 14, Day 14 to 16, Day 14 to 21, Day 14 to29, and Day 14 to 42 and the differences among groups from baseline toDay 14, Day 14 to 16, Day 14 to 21, Day 14 to 29, and Day 14 to 42 willbe assessed. Variables with skewed distributions will be log-transformedbefore analysis. If the data are not normally distributed afterlogarithmic transformation, appropriate nonparametric tests will beused.

The following example and associated results are provided asillustrations of some embodiments of the invention and are not intendedto limit the scope of the claimed subject matter in any way.

EXAMPLES

In the Example below, reference to Compound 1 refers to(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid sodium salt.

Example 1

An experimental study was conducted to evaluate the impact of Compound1, a PPARδ agonist, on muscle atrophy during and following the end oflimb immobilization in human subjects. Except where specifically notedotherwise below, all methods were conducted as described in the protocolabove.

Methods

Methods were generally conducted as described in the protocol. The studywas designed as a double-blind, randomized, placebo-controlled, parallelstudy in healthy male subjects. A total of N=24 subjects wererandomized. The number of randomized subjects is lower than originallyplanned. A total of N=21 subjects had data available for statisticalanalysis. Despite statistical power below that typically used,statistically significant superiority over placebo was observed for theprimary muscle strength variable of analysis.

The study randomized 24 subjects, 12 of whom were randomized to receiveCompound 1 (mean age=42; 50% Black or African American) and 12 of whomwere randomized to receive placebo (mean age=39; 58% Black or AfricanAmerican). One subject had no data post-baseline, and therefore did notprovide usable data. Two other subjects also did not provide usabledata.

Statistical analysis compared the experimental group treated withCompound 1 (n=10) with the placebo group (n=1). Dropout rates in thestudy were 17% in each treatment group.

During an unblinded interim analysis, it was revealed that three of theplacebo-treated subjects increased in muscle strength and also in musclevolume during the immobilization period. Subsequently, a blinded datareview included identification of any subject (regardless of treatmentgroup) who had a significant increase during immobilization in musclevolume as measured by MRI, where a significant increase was defined asan increase of more than 1 standard deviation. A total of 4 subjects metthis criterion, one of which was treated with Compound 1. The analysisconclusions, therefore, are considered to be conservative.

Multiple imputation statistical methods were used to preserve theintent-to-treat (ITT) principle while coping with invalid and missingdata.

Primary analysis was executed as defined in the study protocol and thestatistical analysis plan (see protocol described above). Supportiveanalysis included coping with missing data in different ways to ensurerobustness of analysis conclusions against the methodology used. Methodsincluded: (1) primary (multiple imputation based on recursiveregression), (2) observed cases (no data suppression, no dataimputation), (3) completers (stable group for longitudinal analysis, (4)ITT, last-observation-carried-forward (stable group, all patients), (5)per-protocol set per ICH E9 (excludes protocol violators), (6) use ofplacebo median imputed for missing data, and (7) use of placebo meanimputed for missing data. Analysis results indicated firm robustnessagainst methodology.

Statistical analysis used analysis of covariance (ANCOVA) with baselinemeasure used as a covariate. The least-squares mean (LSMEAN) change is amean change from baseline estimated from the ANCOVA model that reflectsadjustment for baseline values.

Results

FIG. 1 shows a graph of mean changes from baseline in muscle strengthrepresenting the primary analysis (reflecting multiple imputation formissing and invalid data) of the effect of administration of Compound 1on performance of a repeated measures knee extension strength testduring (day 0 to day 14) and after (following day 14) limbimmobilization in human subjects.

The data supporting the graph shown in FIG. 1 is also provided in Table1 below.

TABLE 1 Compound 1 Placebo Mean P-value Time Statistic (n = 10) (n = 11)Difference (2-sample t-test) Baseline Mean 195 176 19 0.3 Day 14 LSMeanchange −2.9 −38.9 36 0.012 Day 21 LSMean change 36.8 −0.3 36.5 0.004(primary) Day 29 LSMean change 30.4 8.5 21.9 0.2

FIG. 2 shows a graph of mean changes from baseline in muscle strengthrepresenting supportive analysis (using all available data for subjectswith valid data, excluding protocol violators, i.e., no imputation) ofthe effect of administration of Compound 1 on performance of a repeatedmeasures knee extension strength test during (day 0 to day 14) and after(day 14 to day 21 and day 21 to day 29) limb immobilization in humansubjects.

The data supporting the graph shown in FIG. 2 is also provided in Table2 below.

TABLE 2 Compound 1 Placebo Mean P-value Time Statistic (n = 9) (n = 8)Difference (2-sample t-test) Baseline Mean 191 168 23 0.4 Day 14 LSMeanchange −1.9 −38.2 36.3 0.04 Day 21 LSMean change 31.5 0.2 31.3 0.048(primary) Day 29 LSMean change 32.5 13.1 19.4 0.2

The data set provided in Table 2 and depicted in FIG. 2 was created fromthe data set provided in Table 3. Table 3 includes the raw values ofmaximum muscle strength on the knee extension (KE) as measured in pounds(lbs) in the repeated measures knee extension strength test. The valuesin this data set reflect no calculation, imputation, or derivation ofany kind. D1 is day 1 (baseline, pre-dose), D14 is day 14 (day whenbrace is removed), D21 is day 21 (primary endpoint for the study), andD29 is day 29, which is the final assessment during the treatmentperiod. D42 is day 42, which is a safety, follow-up assessment, whichwas not intended for statistical analysis.

TABLE 3 Day 1 Day 14 Day 21 Day 29 Day 42 Subject Group (Lbs) (Lbs)(Lbs) (Lbs) (Lbs) A Treated 123 120.5 213 213 238 B Treated 260.5 225.5270.5 270.5 270.5 C Treated 168 158 158 150.5 158 D Treated 163 185.5183 183 183 E Treated 160.5 170.5 205.5 230.5 220.5 F Treated 220.5200.5 270.5 270.5 270.5 G Treated 145.5 195 H Treated 215.5 163 225.5235.5 265.5 I Treated 265.5 255.5 270.5 225.5 270.5 J Placebo 120.5 70.5108 183 170.5 K Placebo 270.5 233 258 270.5 265.5 L Placebo 133 73 180.5188 270.5 M Placebo 215.5 100.5 205.5 220.5 240.5 N Placebo 178 170.5145.5 158 163 O Placebo 110.5 108 110.5 110.5 113 P Placebo 158 145.5160.5 155.5 165.5 Q Placebo 158 168 203 195

Compound 1 was effective in reducing muscle atrophy duringimmobilization (i.e., reducing the rate of loss of muscle strengthduring immobilization relative to control subjects that receivedplacebo) and for reducing atrophy following immobilization (i.e.,increasing the rate of return of muscle strength to baseline followingimmobilization relative to control subjects that received placebo).

It was unexpected that a PPARδ agonist would be associated withpreventing muscle atrophy (i.e., reducing the rate of loss of musclestrength during immobilization relative to control subjects thatreceived placebo). Analysis showed that in subjects treated withCompound 1, measures of muscle atrophy that would be expected did notoccur or could not be measured. In other words, there was a significantreduction in the rate of loss of muscle strength during immobilizationin subjects that received Compound 1 relative to control subjects thatreceived placebo. Further, the rate of loss of muscle strength duringimmobilization in subjects that received Compound 1 was reduced toalmost zero, since subjects that received Compound 1 did not show asignificant loss of muscle strength compared to their baselinemeasurements. Analysis also showed that mean change from baseline to day21 in muscle strength for the group treated with Compound 1 showedsuperiority relative to control subjects that received placebo. By 14days following the end of immobilization (i.e., day 28), change inmuscle strength compared to baseline was no longer significantlydifferent between the group treated with Compound 1 relative to controlsubjects that received placebo.

We claim:
 1. A method of treatment comprising administering to a humansubject(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof, wherein the humansubject has an immobilized limb, and wherein the amount of(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof administered issufficient to reduce the rate of loss of muscle strength in the muscletissue of the immobilized limb of the subject relative to a control,wherein muscle strength is measured by a muscle strength test.
 2. Themethod of claim 1, wherein the rate of loss of muscle strength comprisesa comparison of muscle strength in the subject's immobilized limb to abaseline measurement of muscle strength in the same limb prior to aperiod of immobilization.
 3. The method of claim 1, wherein reducing therate of loss of muscle strength in the subject comprises a return to thesubject's baseline measurement of muscle strength faster than thecontrol following a period of immobilization.
 4. The method of claim 1,wherein reducing the rate of loss of muscle strength in the subjectcomprises a return to the subject's baseline measurement of musclestrength following a period of disuse in less than 90% of the time toreturn to baseline for a control.
 5. The method of claim 1, wherein theloss of muscle strength in the subject's immobilized limb is less thanthe loss of muscle strength relative to the control during a period ofimmobilization.
 6. The method of claim 1, wherein(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof is administeredorally.
 7. A method of treatment comprising administering to a humansubject(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof, wherein the humansubject has an immobilized limb, and wherein the amount of(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof administered issufficient to reduce the rate of loss of muscle mass in the muscletissue of the immobilized limb of the subject relative to a control,wherein the rate of loss of muscle mass comprises a comparison of one ormore measurements of muscle volume in the subject to a baselinemeasurement of muscle volume in the same subject, wherein muscle volumeis measured by the cross-section area of a muscle.
 8. The method ofclaim 7, wherein reducing the rate of loss of muscle mass in thesubject's immobilized limb comprises a return to the subject's baselinemeasurement of muscle mass faster than the control.
 9. The method ofclaim 7, wherein reducing the rate of loss of muscle mass in thesubject's immobilized limb comprises a return to the subject's baselinemeasurement of muscle mass following a period of limb immobilization inless than 90% of the time to return to baseline for a control.
 10. Themethod of claim 7, wherein the loss of muscle mass in the subject isless than the loss of muscle mass relative to the control.
 11. Themethod of claim 7, wherein the loss of muscle mass in the subject'simmobilized limb comprises less than a 10% loss of muscle mass relativeto the subject's baseline measurement of muscle mass prior to a periodof immobilization.
 12. The method of claim 7, wherein(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof is administeredorally.
 13. A method of treatment comprising administering to a humansubject(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof, wherein the humansubject has an immobilized limb, and wherein the amount of(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof administered issufficient to reduce the rate of loss of Type I muscle fibers inimmobilized muscle tissue of the subject relative to a control, whereinthe measure of the rate of loss of Type I muscle fibers in theimmobilized muscle tissue comprises a comparison of one or moremeasurements of Type I muscle fibers in the subject to a baselinemeasurement of Type I muscle fibers in the same subject.
 14. The methodof claim 13, wherein(E)-[4-[3-(4-Fluorophenyl)-3-[4-[3-(morpholin-4-yl)propynyl]phenyl]allyloxy]-2-methyl-phenoxy]aceticacid or a pharmaceutically acceptable salt thereof is administeredorally.